Mycobacterium tuberculosis, the agent of tuberculosis, is the leading cause of death in adults worldwide (14). The emergence of drug resistant strains (48) and the problems associated with tuberculosis in HIV-infected populations (18) have brought tuberculosis research to the forefront. The development of genetic techniques to study the biology of the organism is an important goal of mycobacterial research.
Considerable effort has gone into the development of allelic exchange methods to selectively disrupt genes of various mycobacterial species. Several groups have used either small linear DNA fragments (4, 25, 43), long linear DNA fragments (5), or suicidal plasmids, (37, 44) (9, 27, 39, 41, 42) to achieve allelic exchange in both fast and slow-growing mycobacteria. Slow-growing mycobacteria such as M. tuberculosis and M. bovis BCG can integrate exogenous DNA into their chromosome by both illegitimate and homologous recombination (2, 25). Allelic exchange in fast-growing mycobacteria such as M. smegmatis is easier than in the slow-growing species; this has led to the idea that the homologous recombination machinery of slow-growing mycobacteria is rather inefficient (32).
Thus far, the only mutants constructed in the slow-growing mycobacterial species are those with genes disrupted with an antibiotic marker. However, in many cases an antibiotic marker may not be desirable. It may not be known whether or not a gene is essential and targeted disruption does not let one ascertain essentiality. The failure to obtain a mutant might be due to the failure of the methodology and not to the essentiality of the gene. Furthermore, the possibility of polar effects from an inserted antibiotic marker can prevent the disruption of a non-essential gene if that gene is located in an operon upstream of an essential gene. Also, there are a limited number of antibiotic resistance genes available for use in mycobacteria and making a marked mutation excludes one antibiotic from further consideration. In addition, mutants that are potential vaccine candidates should not contain antibiotic resistance determinants.
An ideal allelic exchange system is one that can be used for the exchange of unmarked deletion alleles as well as alleles with point mutations. Constructing knockout mutants by in-frame deletions would negate the concerns with using a targeted disruption method. Such mutants are antibiotic sensitive, cannot revert, and the mutations should not be polar on the expression of downstream genes. By extension, the same technique could be used for allelic exchange of point mutations, allowing for a finer dissection of gene function. This allelic exchange methodology, utilizing a plasmid unable to replicate in the organism of interest and selectable and counter-selectable markers (15), has been successfully used in M. smegmatis (27, 41). The inventors sought to determine if such an allelic exchange methodology would reproducibly work for the slow-growing mycobacteria, such as M. bovis BCG and M. tuberculosis. 
The inventors describe herein a new mycobacterial suicide plasmid for allelic exchange of unmarked mutations utilizing sacB sucrose counter selection. This counter selectable marker was previously reported to work in mycobacteria, including M. tuberculosis and M. bovis BCG (40) (42) (9). However, the previously described mycobacterial sacB vector systems were used for allelic exchange of genes disrupted with an antibiotic resistance marker. The present invention demonstrates the reproducibility of this system for allelic exchange of unmarked deletions in the chromosome of M. smegmatis, M. bovis BCG and M. tuberculosis. The inventors have also constructed lysine auxotrophs of these three organisms by allelic exchange of lysA, the gene encoding meso-diaminopimelate decarboxylase, the last enzyme in the lysine biosynthetic pathway (52). To the best of the inventors"" knowledge, this is the first report of the construction of unmarked deletion mutations in the genome of slow-growing mycobacteria.
The present invention discloses a slow-growing recombinant mutant mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation. The invention further provides a method for preparing the recombinant mutant mycobacterium of the present invention comprising introducing a vector into a slow-growing mycobacterium, where said vector comprises a selectable marker, a counter selectable marker, and an unmarked mutant mycobacterial gene, culturing the slow-growing mycobacterium and selecting for primary recombinants incorporating the selectable marker. The primary recombinants are then cultured, and secondary recombinants that have lost the counter selectable marker are selected for, followed by isolation of the secondary recombinants incorporating the desired unmarked mutant mycobacterial gene.
Also provided is a vaccine comprising the slow-growing recombinant mutant mycobacterium of the present invention contained in a physiologically acceptable carrier, as well as a method of treating or preventing tuberculosis in a subject comprising administering the vaccine of the present invention in an amount effective to treat or prevent tuberculosis in the subject.